Methods of administering camptothecin compounds for the treatment of cancer with reduced side effects

ABSTRACT

Methods of administering camptothecin compounds such as irinotecan hydrochloride to reduce a diarrhea side effect and methods of treating cancer and AIDs with camptothecin compounds including administering the camptothecin compounds while maintaining the intestinal lumen and the bile at an alkaline pH.

FIELD OF THE INVENTION

[0001] The present invention relates to camptothecin compounds, inparticular, irinotecan hydrochloride composition formulations, andmethods of administering camptothecin compounds such as irinotecanhydrochloride for the treatment of cancer and AIDS, with reduced sideeffects.

BACKGROUND OF THE INVENTION

[0002] Camptothecin is a quinoline-based alkaloid found in the barks ofthe Chinese Camptotheca tree and the Asian nothapodytes tree. It is aclose chemical relative to aminocamptothecin, CPT-11 (irinotecan),DX-8951F and topotecan. These compounds are useful in treating breastcancers, ovarian cancer, colon cancer, malignant melanoma, small celllung cancer, thyroid cancers, lymphomas and leukemias. These compoundsare also used for the treatment of AIDS.

[0003] Irinotecan hydrochloride (CPT-11)(4S)-4,11-diethyl-4-hydroxy-9-[(4-piperidinopiperidino)carbonyloxy]1H-pyrano[3′, 4′:6,7] indolizino,[1,2-b]quinoline-3,14(4 h,12H)dionehydrochloride, has a novel mechanism of antitumor activity, namely theinhibition of DNA topoisomerase I. Topoisomer-ases are the enzymes whichwind and unwind the DNA that makes up the chromosomes. As thechromosomes must be unwound to make proteins, camptothecin compoundskeep the chromosomes wound tight so that they cannot make proteins.Because cancer cells grow at a much faster rate than normal cells, theyare more vulnerable to topoisomerase inhibition than normal cells.

[0004] CPT-11 has shown effective antitumor activity clinically (2, 3),and, recently, a survival benefit by CPT-11 was shown in colorectalcancer. However, it has major toxicities of leukopenia and diarrhea inclinical practice. The clinical use of CPT-11 at higher dosages wasassociated with an unexpected and significant incidence of diarrhea (4,6, 7, 12), and diarrhea is now recognized as a dose-limiting toxicity ofthis drug (4-7). Although many pharmacokinetic analyses, which haveshown a great interpatient variability, have been made to predict theincidence of diarrhea, there are somewhat conflicting results (8-11).

[0005] CPT-11 and its metabolites, SN-38 and SN-38-Glu, were detected innot only human plasma but also human bile. Of the three compounds, SN-38has strong cytotoxicity, SN-38-Glu is a deactivated, glucuronidated formof SN-38, and CPT-11 has much less cytotoxicity compared to SN-38. Thesecompounds have an α-hydroxy-3-lactone ring, which undergoes reversiblehydrolysis at a rate that depends mainly on pH (15, 16, 17). At morethan physiological pH, the lactone form is unstable and the equilibriumfavors hydrolysis to open the lactone ring and yield the carboxylateform. Under acidic conditions, the reverse reaction, with formation ofthe lactone, is favored. Similar reactions also occur with CPT-11 andSN-38-Glu.

[0006] From several reports, it is considered that major metabolicpathways in human are as follows; CPT-11 is hydrolyzed bycarboxylesterase of mainly liver origin to the active metabolite,7-ethyl-10-hydroxy-camptothecin (SN-38). Some of SN-38 undergoessubsequent conjugation by the hepatic enzyme, UDP-glucuronyltransferase,to SN-38 β-glucuronide (SN-38-Glu), and is excreted into bile along withthe other components, CPT-11 and SN-38 (13, 14). The three compounds arebelieved to be reabsorbed by intestinal cells to enter the enterohepaticcirculation. Recently, it has been found that hepatic cytochrome P-4503A enzymes metabolize CPT-11 to 7-ethyl-10-[4-N-(5-aminopentanoicacid)-1-piperidino]carbonyloxycamptothecin, which has 500-fold weakerantitumor activity than SN-38 (Rivory et al., 1996b; Haaz et al., 1997).CPT-11, SN-38 and SN38-Glu have an α-hydroxy-3-lactone ring, whichundergoes reversible hydrolysis at a rate which is mainly pH-dependent(Fassberg et al., 1992). At physiological pH and higher, the lactoneform is unstable and the equilibrium favors hydrolysis to open thelactone ring and yield the carboxylate form. Under acidic conditions,lactone-carboxylate interconversion is shifted toward the lactone form.CPT-11, SN-38 and SN38-Glu are excreted into bile and along with it arereleased into the small intestinal lumen (Atsumi et al., 1991; Lokiec etal., 1995; Chu et al., 1997a, b). Furthermore, although minor (Atsumi etal 1995), an additional pathway involves direct transport of CPT-11 andits metabolites from serum to lumen across the intestinal epithelialcells. Once in the intestine, SN38-Glu can be deconjugated in the cecumand colon to SN-38 by bacterial β-glucuronidase (Takatsuna et al.,1996). CPT-11, SN-38 and SN38-Glu are believed to be reabsorbed to acertain extent by intestinal cells and to enter the enterohepaticcirculation.

[0007] To date, there is little information about the intestinal uptakeand transport mechanism of CPT-11 and its derivatives. This knowledge isa critical step in the understanding of the mechanism by which CPT-11induces diarrhea. In the present study, the uptake of CPT-11 and SN-38by intestinal epithelial cells was estimated and correlated to theirrespective effect on cell toxicity.

[0008] The structure of several camptothecin derivatives are known.

[0009] In addition, U.S. Pat. No. 5,552,154 discloses that camptothecin(CPT) and derivatives thereof of the closed lactone ring form areadministered intramuscularly or orally. In such cases, it was possibleto obtain total remissions of a vast spectrum of human cancers withoutthe toxicity observed previously with CPT Na+. The derivatives of CPTused were 9-Amino-20 (S)-Camptothecin (9AC). 9-Nitro-20(S)-Camptothecin(9NO₂).

[0010] U.S. Pat. No. 5,468,754 describes that CPT 11 and othercamptothecin derivatives undergo an alkaline, pH-dependent hydrolysis ofthe E-ring lactone. The slow reaction kinetics allow one to assesswhether both lactone and non-lactone forms of the drug stabilize thetopoisomerase-cleaved DNA complex. Studies indicate that only the closedlactone form of camptothecin helps stabilize the cleavable complex.Therefore, the patent recommends that pH levels of below 7 be used toallow the lactone form of camptothecin to predominate. The patentsuggests the administration of the compounds with a pharmaceuticallyacceptable acid.

[0011] U.S. Pat. No. 5,447,936 describes that the HECPT form of the drugis more effective in inhibiting topoisomerase-I in an acidicenvironment, than in cells having higher intracellular pH levels. Thepatent describes the administration of the drug with an acid which is anorganic carboxylic acid such as citric acid.

[0012] U.S. Pat. No. 5,225,404 describes the administration of acamptothecin compound with water-based solvents for water-solublecompounds such as normal saline or phosphate buffered saline solutions.The patent indicates that signs of diarrhea and cystitis were preventedand no overall toxicity was obtained.

[0013] U.S. Pat. No. 5,637,770 describes the creation of a hexacycliccompound obtained by the addition of a water-soluble ring tocamptothecin, which had superior characteristics to camptothecin. U.S.Pat. No. 5,633,016 describes a combination cancer therapy includingadministering an effective amount of topotecan with cisplatin.

[0014] U.S. Pat. No. 5,633,260 discloses a 7-11-substituted camptothecinderivative. The patent also describes that maintaining an acidic pH (3to 4) in the formulation is important to reduce the slow conversion of11,7-HECPT lactones with the E-ring-hydrolyzed carboxylate which occursat physiological pH. This patent prescribes regulated dosages toeliminate toxicity of the compound.

[0015] U.S. Pat. No. 5,652,244 describes a method of treating humancarcinoma with camptothecin derivatives. U.S. Pat. No. 5,658,920describes a hexacyclic compound derivative of camptothecin.

[0016] U.S. Pat. No. 5,597,829 discloses that CPT is excreted unchangedby the kidneys, although a large percentage of the drug administeredcannot be accounted for in the urine. The patent suggests that enhancedrenal excretion of the carboxylate form of CPT occurs when exposed to apH lower than 5. Therefore, it is recommended the administration of thedrug to assure an acidic pH value by administering the compound withorganic carboxylic acids.

[0017] U.S. Pat. No. 5,674,874 describes the pharmacologic conversion ofCPT 11 to HECPT. The patent describes administration of the compound insufficient quantities to maintain the pH of the formulation from about 2to about 6 with the administration of a pharmaceutically acceptableacid.

[0018] Cancer Investigation, Volume 14, Supplement 1, No. 31, describesthe use of irinotecan (CPT 11) to treat colon cancer and non-smallcellular lung cancer. The publication confirms the incidence of grade 4diarrhea associated with administration of CPT 11 dropped from 17% to 5%following adoption of an aggressive loperimide therapy.

[0019] Irinotecan Approved for Advance Colorectal Cancer, Med. Sci. Bull1996; Volume 18, No. 12, describes that diarrhea is a common side effectof irinotecan administration.

[0020] Journal of the National Cancer Institute, September 4, 1996, Vol.88, No. 17, suggests that excessive production of sulphomucin in thececum could be the major cause of CPT-11-induced diarrhea.

[0021] The Camptosar Patient Management Guidelines suggest avoiding thediarrhea side effect of camptosar by administering loperimides andgatorade.

[0022] The present invention overcomes one of the major side effects,diarrhea, associated with administration of camptothecin compounds, inparticular irinotecan hydrochloride. This is one of the majordeficiencies in the prior art in delivering irinotecan hydrochloride forthe treatment of tumors. The present invention overcomes the diarrheaside effect associated with the administration of irinotecanhydrochloride and its related compounds.

SUMMARY OF THE INVENTION

[0023] The present invention provide for methods of administeringcamptothecin compounds which are cleared through the liver, preferablyirinotecan hydrochloride and its derivatives.

[0024] The invention provides a method of inhibiting a diarrhea sideeffect of camptothecin compounds cleared by the liver, including but notlimited to, irinotecan hydrochloride (CPT-11), SN38-Glu, and SN-38comprising administering irinotecan hydrochloride while the intestinallumen is maintained an alkaline pH.

[0025] The invention also provides a method of treating cancercomprising administering camptothecin compounds such as irinotecanhydrochloride while maintaining the intestinal lumen at an alkaline pH.

[0026] In a preferred embodiment the cancer is selected from, but notlimited to, breast cancer, ovarian cancer, colon cancer, malignantmelanoma, small cell lung cancer, thyroid cancers, lymphomas andleukemias.

[0027] In another embodiment the invention provides a method of treatingAIDS comprising administering irinotecan hydrochloride while maintainingthe intestinal lumen at an alkaline pH.

[0028] The invention advantageously provides a method of administering acamptothecin compound such as irinotecan hydrochloride (CPT-11)intravenously comprising prior to or simultaneously administering saidcamptothecin compound, orally administering a bicarbonate and alkalineH₂O.

[0029] The invention provides a method of administering a camptothecincompound such as irinotecan hydrochloride (CPT-11) intravenouslycomprising prior to or simultaneously administering said camptothecincompound, orally administering a composition comprising borbic acid.

[0030] The invention also provides for a method of administering acamptothecin compound comprising prior to or simultaneouslyadministering said camptothecin compound, orally administering acomposition comprising urso-deoxycholic acid.

[0031] Throughout the present specification where compositions, kits,and methods are described as including or comprising specificcomponents, it is contemplated by the inventors that compositions of thepresent invention also consist essentially of or consist of the recitedcomponents.

[0032] The above and other objects of the invention will become readilyapparent to those of skill in the relevant art from the followingdetailed description and figures, wherein only the preferred embodimentsof the invention are shown and described, simply by way of illustrationof the best mode of carrying out the invention. As is readily recognizedthe invention is capable of modifications within the skill of therelevant art without departing from the spirit and scope of theinvention.

BRIEF DESCRIPTION OF THE FIGURES

[0033]FIG. 1 shows structures of CPT-11, SN-38 and SN-38-glucuronide(SN-38-GLU): Lactone forms of CPT-11 and SN-38 are non-ion charged, andcarboxylate forms of CPT-11 and SN-38 are anions. Not only carboxylateform of SN-38-Glu but also its lactone form, which possesses anadditional carboxyl group in its glucuronide moiety, is an anion. Thereversible conversion between lactone and carboxylate forms is pHdriven.

[0034]FIGS. 2A and 2B show the time course of CPT-11 and Sn-38 uptake byisolated intestinal cells: The uptake of [¹⁴C] CPT-11 (20 μM) and [¹⁴C]SN-38 (2 μM) in lactone and carboxylate form, respectively, by isolatedintestinal cells from jejunum was measured as a function of time. Attime 0, the respective agent was added to the intestinal cell suspensionmaintained at 37° C. under permanent shaking. At 15, 30, 45, 60, 90,120, 240 and 480 sec, aliquots of cell suspension were removed, andprocessed as described in Materials and Methods. The results shown aremean ±SE of n experiments.

[0035]FIGS. 3A, 3B, 3C and 3D show the relationship between initial rateof uptake of CPT-11 and its concentration: The initial uptake rate wasdetermined from the linear slope of the cellular uptake over the initial90 sec incubation period. The data were fitted by least-square nonlinearregression analysis using the equation V=(V_(max) S)/(K_(m)+S)+K_(d) Swhere V represents the initial rate of uptake, V_(max) is the maximumrate of uptake, K_(m) is the apparent Michaelis constant, K_(d) is therate of diffusion and S is the concentration of CPT-11.

[0036]FIGS. 4A and 4B show the relationship between initial rate ofuptake of SN-38 and its concentration. The initial uptake rate wasdetermined as described in legend of FIG. 3 and in Materials andMethods. The data were fitted by least-square linear regression. Becauseof limited solubility, only concentrations of SN-38 up to 2 μM wereinvestigated.

[0037]FIG. 5 shows the effect of taurocholate (TCA) on respective CPT-11and SN-38 micelle formation: [¹⁴C] CPT-11 (20 μM) and [¹⁴C] SN-38 (2 μM)were stored overnight in Hank's solution in the presence of absence ofTCA (20 mM). Monomers were separated from micellar aggregates byultrafiltration through a 1000-molecular weight cut-off membrane (YM1)as described in Materials and Methods. Values are the monomeric forms ofthe indicated metabolites, expressed as a percentage (%) of theconcentration of the initial solution before ultrafiltration. Comparisonbetween TCA and control was estimated by Mann-Whitney test. (⁺): Sn-38carboxylate is significantly different from the other agents tested inthe presence of TCA (Kruskal-Wallis test: p=0.023, Student-Newman-Keulsmethod, p<0.05). Abbreviations used: CPT lactone (CPT lact.); CPTcarboxylate (CPT carb.); SN-38 lactone (SN lact.); and SN-38 carboxylate(SN carb.).

[0038]FIGS. 6A and 6B show the effect of pH on the initial rate ofuptake of CPT-11 and SN-38: [¹⁴C] CPT-11 (20 μM) and [¹⁴C] SN-38 (2 μM)were dissolved in PBS at pH 6.2, 6.8, 7.4 and 8.0 and stored overnight.By adding the drugs to Hank's solution containing intestinal cells fromwhole small-intestine, uptake studies were performed. The difference inthe initial uptake rate by pH was analyzed by Kruskal-Wallis test(p<0.001 and p<0.001 for CPT-11 and SN-38, respectively) andStudent-Newman-Keuls method (*p<0.05).

[0039]FIG. 7 shows the effect of pH on the initial uptake rate of HT29cells. [¹⁴C]SN-38 (2 μM) were dissolved in PBS at pH 6.2, 6.8, 7.4 and8.0 overnight. The uptake study was 2p initiated by adding the compoundsto Hanks' solution containing HT29 cells. The comparative initial rateof uptake as function of pH was analyzed by Kruskal-Wallis test(p<0.001) and Dunn's method (*p<0.05).

[0040]FIG. 8 shows the relationship between the initial uptake rate andthe cytotoxicity of SN-38. Using HT29 cells, the effect of physiologicalpH on the initial uptake rate of 2 μM [¹⁴C]SN-38 was estimated asdescribed in legend of FIG. 3. The 0.4 μM SN-38-induced cytotoxicity inHT29 cells was studied by the described MTT assay. The relationshipbetween the initial rate of uptake and the cytotoxicity of SN-38 wasplotted by a simple least-squares regression method.

DESCRIPTION OF THE INVENTION

[0041] Knowledge of the cellular transport mechanism of camptothecincompounds such as CPT-11 and its metabolites by the intestine is acritical step in the understanding of the mechanism by whichcamptothecin compounds, such as CPT-11, induce diarrhea and its greatinterpatient variability in pharmacokinetics. The inventors reviewed theuptake of several camptothecin compounds, CPT-11 and SN-38, byintestinal epithelial cells. The results provide for the new design ofan approach to prevent diarrhea and large interpatient variability inpharmacokinetics in clinical practice of the treatment of cancer andtumors with irinotecan hydrochloride and its related compounds.

[0042] The invention provides a method of inhibiting a diarrhea sideeffect of camptothecin compounds such as irinotecan hydrochloride(CPT-11), SN-38-Glu, SN-38 and its derivatives comprising administeringirinotecan hydrochloride while maintaining the bile and/or intestinallumen at an alkaline pH. In a preferred embodiment the intestinal lumenis maintained at an alkaline pH by administration of bicarbonate andalkaline H₂O. The amount of bicarbonate and alkaline pH is suitable toreduce the uptake of the camptothecin compound and thus reduce thecytotoxic side effects including a diarrhea side effect. Thecamptothecin compound or irinotecan hydrochloride may be administeredintravenously, orally or intramuscularly. The method of the inventioninhibits the reabsorption and decreases the lactone uptake of CPT-11 andSN-38 by the intestines and thus reduces the diarrhea side effectassociated with camptothecin compounds such as irinotecan hydrochloride.

[0043] The invention also provides a method of treating cancercomprising administering irinotecan hydrochloride and its derivatives ormixtures thereof, while maintaining the intestinal lumen at an alkalinepH. In a preferred embodiment the cancer is selected from the groupconsisting of, but not limited to breast cancer, ovarian cancer, coloncancer, malignant melanoma, small cell lung cancer, thyroid cancers,lymphomas and leukemias. The alkaline pH may be a pH from about 7 toabout 10. In an alternative embodiment the cancer is treated byadministering a compound selected from 7-hydroxymethyl camptothecin,irinotecan hydrochloride, aminocamptothecin, DX-8951F, SN-38, HAR4,HAR5, HAR6, HAR7, HAR8 and topotecan, while maintaining the intestinallumen at an alkaline pH.

[0044] The invention advantageously provides for a method of treatingAIDS comprising administering irinotecan hydrochloride or itsderivatives while maintaining the intestinal lumen at an alkaline pH.

[0045] A pharmaceutical composition and kit including irinotecanhydrochloride (CPT-11) administered in combination with a bicarbonateselected from sodium bicarbonate, magnesium bicarbonate and potassiumbicarbonate. Alternatively irinotecan hydrochloride (CPT-11) may beadministered in combination with a composition comprising borbic acid.This chemical has been used in buffers composition, such as theBritton-Robinson buffer and has a strong alkalinic buffering action.

[0046] The invention also provides for a method of administering acamptothecin compound comprising prior to or simultaneouslyadministering said camptothecin compound, orally administering acomposition comprising urso-deoxycholic acid. This composition mayoptionally be administered with bicarbonate. It is believed thaturso-deoxycholic acid stimulates bicarbonate secretion into bile.

[0047] The following example shows the ability to reduce the diarrheaside effect of irinotecan hydrochloride compounds in accordance with themethod of the invention.

EXAMPLE

[0048] Drugs and Animals

[0049]¹⁴C-Labeled SN-38 (3.68 MBg/mg) and ¹⁴C-Labeled CPT-11 (1.47MBq/mg) were kindly donated by Daiichi Pharmaceutical Co., Ltd. Tokyo,Japan). Non-labeled CPT-11, SN-38, and SN-38-Glu were supplied by YakultHonsha Co., Ltd. Tokyo, Japan). ¹⁴C-labeled SN-38 was dissolved in DMSOat a final concentration 2 μM because it was very hydrophobic and poorlysoluble in water. DMSO at 2% was confirmed to have no effect on theinitial uptake of labeled CPT-11 and SN-38. The other drugs weredissolved in distilled water. The lactone and carboxylate forms of¹⁴C-labeled CPT-11 and SN-38 were produced by dissolving the compoundovernight in 50 mM phosphate buffer at pH 6. or 9, respectively. DNP-SGwas made from glutathione and CDNB (1-chloro-2,4-dinitro benzene)chemically. All other reagents were of analytical grade. Adult maleGolden Syrian hamsters (6-8 weeks age), whose model presents a bile acidprofile similar to that observed in human (28), were used.

[0050] Preparation of Intestinal Cells

[0051] Intestinal cells were isolated as previously described (28, 29).Briefly, male hamsters were anesthetized with sodium pentobarbital(Nembutol 70 mg/kg body weight). The entire intestine was removed. Theintestinal lumen was washed with 37° C. Hank's solution. Sacs of theileum (12.5 cm from cecum) and jejunum (remaining small intestine) wererinsed, as well as the intestinal sacs of the anal site of smallintestine (12.5 cm from cecum) and oral site (the other smallintestine). The sacs were rinsed with oxygenated buffer solutioncontaining sodium citrate (96 mM NaCl, 1.5 mM KCl, 5.6 mM KH₂PO₄, 27 mMsodium citrate, pH 7.3), and incubated for 10 min in the same buffer at37° C. The sacs were then emptied, filled with oxygenated buffersolution containing EDTA (140 mM NaCl, 16 mM Na₂HPO₄, 2 mM EDTA, 0.5 mMdithiothreitol, pH 7.3), incubated for 10 min at 37° C. Then each sacwas placed onto a petri dish and gently vortexed for 1 min. The buffercontaining intestinal cells was recovered in 50 mL of Hanks' solution,washed twice and adjusted at 10⁶ cells/ml in Hanks' medium (cellularstock solution containing 0.5% bovine serum albumin, pH 7.4)..

[0052] Determination of the Cellular Uptake of ¹⁴C-labeled CPT-11 andSN-38, respectively

[0053] Uptake of ¹⁴C-labeled CPT-11 and SN-38 was measured by rapidvacuum filtration assay (28, 29). The cellular suspension of 0.95 ml wasincubated for 15 min in a 37° C. water bath with stirring. Uptake wasstarted by the addition of 0.05 ml PBS (at pH 3 or 9) containing labeledSN-38 or CPT-11 at 37° C. At various time intervals, 100 μL, samplealiquots were diluted into 3 mL of Hank's medium at 4° C. to stop theuptake. The stop solution containing the cells was filtered through aglass microfiber filter (Glass Fiber Filter Circles G4, Fisherbrand,Pa.) under vacuum (20 psi). The cells were washed once with 5 mL of 0.5%bovine serum albumin-containing Hanks' medium (4° C.) and once with 20mL of Hanks' solution (4° C.). The filters were placed in a vialcontaining 4 mL of scintillation liquid (Ultra Gold, Packard, Conn.) andthe radioactivity was counted in a β scintillation counter (LS3801,Beckman, Md.).

[0054] The effect of the metabolic inhibitor, 2,4 dinitrophenol (1 mM),was studied by adding this agent to the cells 3 min prior to either¹⁴C-CPT-11 or ¹⁴C-SN-38 (2 μM). The effect of 20 mM of taurocholic acid(TCA) on the uptake of both CPT-11 and SN-38 was investigated followingovernight incubation of ¹⁴C-CPT-11 (20 μM) and ¹⁴C-SN-38 (2 μM) inHank's solution, at pH 7.4 and in the presence and absence of TCA. Theeffect of 200 μM of DNP-SG or SN-38-Glu was also studied by adding theseagents to the cell preparation 7 min prior to either ¹⁴C-CPT-11 (20 μM)and ¹⁴C-SN-38 (2 μM).

[0055] The effect of physiologic pH on the initial intestinal uptakerate of CPT-11 and SN-38 was investigated following overnight incubationof ¹⁴C-CPT-11 (20 μM) and ¹⁴C-SN-38 (2 μM) in phosphate buffered salineat pH 6.2, 6.8, 7.4 and 8, respectively.

[0056] Estimation of Micelle Formation

[0057] To assess whether or not CPT-11 and SN-38 form micelles, theseagents were incubated overnight at pH 4 and 9 in a calcium and magnesiumfree Hank's solution containing 10 mM TCA. The respective solution wasfiltered through a 1000-molecular weight cut-off membrane YM1 (Diaflo,Amicon, Mass.) at a steady speed of 0.04 ml/min. Once the filtration wasstopped, the radioactivity in the initial solution as well as in thefiltrate and in the retained solution after filtration was determined asdescribed previously.

[0058] Cytotoxicity Assay

[0059] Rapid colorimetric assay for mitochondrial dehydrogenase activitywas modified and used for the estimation of cytotoxicity of SN-38(Mosmann, 1983). Briefly, HT29 cells were seeded into a 12-well plate(Falcon-3043, Lincoln Park, N.J.), and, after 48 h, SN-38 (0.4 μM) at pH6.2, 6.8, 7.4 and 8.0 was added. After 24 h-exposure, the cells werewashed twice, and subjected to a drug-free incubation for 24 h. Then,the cells were incubated with 0.5 mg/ml3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) for4 h, and the blue formazan crystals were solubilized by addition of 10%n-dodecylsulfate sodium salt (SDS) in 0.1N HCl and overnight incubation.The formation of the blue formazan compound is spectrophotometricallydetermined at 560 nm (Ultraspec 4050, LKB, Bromma, Sweden).

[0060] Statistical Analysis

[0061] The initial rate of uptake of CPT-11 or SN-38 was derived fromthe linear regression analysis of the respective regression lineobtained from the plot of the uptake as a function of time. The initialrates of uptake were plotted against the corresponding concentration.The data were fitted by least-squares nonlinear regression analysis(SigmaStat, Jandel Scientific, Calif.), using the equationV=(V_(max)·S)/(K_(m)+S)+K_(d)·S where V represents the initial rate ofuptake. V_(max) is the maximum rate of uptake, K_(m) is the apparentMichaelis constant, K_(d) is the rate of diffusion and S is theconcentration of CPT-11 or SN-38.

[0062] Comparisons between two groups were evaluated by the Mann-WhitneyRank Sum Test. Statistical significance of differences among more thantwo groups was determined by Kruskal-Wallis One Way Analysis of Varianceon Ranks, then multiple comparisons versus control group were performedby Dunn's Method. The correlation between the initial rate of uptake andthe cytotoxicity of SN-38 was plotted by a simple least-squaresregression method.

[0063] Uptake of CPT-11 and SN-38 Lactone and Carboxylate, Respectivelyby Intestinal Cells

[0064] The time-dependent uptake of 20 μM ¹⁴C-CPT-11 and 2 μM ¹⁴C-SN-38in both lactone and carboxylate forms by isolated jejunal cells is shownin FIG. 2. The extrapolation of the uptake value at time 0 yields apositive intercept, indicative of non-specific binding, such asadsorption to labeled agents on the cell surface. The respective uptakeof the lactone and carboxylate forms of both CPT-11 and SN-38 was linearfor up to 90 sec. Therefore, the initial uptake rate was determined bylinear regression fit of the uptake over the initial period of time.Comparison of the uptake rate between the lactone and carboxylate formof the respective agent clearly showed a more rapid uptake of bothCPT-11 and SN-38 lactone, as compared to carboxylate form (FIG. 2).

[0065] Table 1 summarizes the respective initial uptake rate of 20 μM¹⁴C-CPT-11 and 2 μM ¹⁴C-SN-38 by jejunal and ileal cells. CPT-11 andSN-38 lactone were more rapidly taken up than their carboxylate forms incells from both intestinal regions but without significant differencesbetween jejunal and ileal cells.

[0066] Transport System of CPT-11 and SN-38 Lactone and Carboxylate

[0067] The respective initial uptake rate of CPT-11 lactone andcarboxylate was plotted as a function of the concentration and the datafor were fitted by least-squares nonlinear regression analysis using theequation V=(V_(max)·S)/(K_(m)+S)+K_(d)·S (FIG. 3) in both jejunal andileal cells, the predominant component of the uptake of CPT-11 lactonewas non-saturable, suggesting uptake by either passive diffusion orfluid-phase endocytosis. The analysis of the curve of the uptake ofCPT-11 carboxylate suggested also at least two separate components ofthe uptake process. The saturable component of the curve wascharacterized by a maximum rate of uptake (V_(max)) of 147 and 157pmol·10⁶ cell⁻¹·min⁻¹·μM⁻¹ and a Michaelis constant (K_(m)) of 51.3 and50.5 μM in jejunal and ileal cells, respectively. The minornon-saturable component was characterized by a diffusion constant(K_(d))<0.05 pmol·10⁶ cell⁻¹·min⁻¹·μM⁻¹ and represented less than onetwentieth of that for CPT-11 lactone in cells of both intestinal regions(Table 2). Furthermore, the Kd for CPT-11 lactone was 1.8-2.5 fold lowerthan that of SN-38 lactone.

[0068] The initial uptake rate of SN-38 lactone and carboxylate wasplotted as a function of the concentration (FIG. 4). The maximumconcentration of SN-38 used in this study was lower than 2 μM due to thepoor solubility of the compound and therefore rendered the determinationof the saturable and unsaturable component of the uptake difficult. Inthis range of concentrations, the uptake of SN-38 lactone andcarboxylate was mostly non-saturable (FIG. 4).

[0069] The carrier-mediated transport is known to be inhibited bymetabolic poisons, such as 2,4-dinitrophenol, which interferes with cellmetabolism and reduces energy-producing reactions (23). Therefore,2,4-dinitrophenol was used in applicants study to determine themechanism of uptake of CPT-11 and SN-38 lactone and carboxylate,respectively. The results of this study are summarized in Table 3.Although, the uptake rate of both CPT-11 and SN-38 lactone was notsignificantly affected by the addition of 2,4-dinitrophenol, the uptakerate of CPT-11 and SN-38 carboxylate was reduced to 22.6 and 30.8%,respectively by 2,4-dinitrophenol, suggesting an active transportmechanism for both of these compounds.

[0070] 2,4-dinitrophenol-S-glutathione (DNP-SG) is known to be asubstrate for the active multispecific organic anion transporter (cMOAT)in the liver (24). In addition, the conjugation byUDP-glucuronyltransferase of SN-38 leads to the formation of SN-38-Gluwhich is also a substrate for the hepatic CMOAT (12,17). To determinewhether either CPT-11 carboxylate and/or SN-38 carboxylate istransported through a cMOAT-like mechanism in intestinal cells, theuptake rate of CPT-11 and SN-38 was studied in the presence or absenceof both DNP-SG and SN-38-Glu. The results are summarized in Table 3.DNP-SG and SN-38-Glu significantly inhibited the the uptake of thecarboxylate form of SN-38 by over 60% while that of CPT-11 carboxylateremained unchanged. The uptake rates of the lactone forms of CPT-11 andSN-38 were not significantly affected by the presence of either DNP-SGor SN38-Glu.

[0071] Micelle Formation and its Effect on the Initial Uptake Rate ofCPT-11 and SN-38

[0072] Taurocholic acid (TCA) at a concentration greater than itscritical micellar concentration forms micelles (25) which in contrast toboth CPT-11 and SN-38, cannot pass through a 1,000-molecular weightcut-off membrane. We used this property to determine whether or notCPT-11 and SN-38 lactone and carboxylate can associate to the TCAmicelles. The results reported in FIG. 5, show that TCA significantlydecreased the % monomer concentration of CPT-11 lactone and carboxylateas well as SN-38 lactone. However, SN-38 carboxylate did notsignificantly associate to TCA micelles.

[0073] Next applicants tested the effect of micelle formation on thecellular uptake of CPT-11 and SN-38. In this series of experiments, thecells from the jejunum and ileum were combined. In the presence of 20 mMTCA, the initial uptake rate (mean±SD) of CPT-11 and SN-38 was reducedto 48.5±10.8 and 69.3±12.7% of control without TCA, respectively (n=5,Mann-Whitney test, p=0.015 and p=0.343 for CPT-11 and SN-38,respectively).

[0074] Effect of pH and Bicarbonate on the Initial Uptake Rate of CPT-11and SN-38

[0075] The interconversion between the lactone and carboxylate CPT-11and SN-38, respectively, is reversible and pH-driven (11). The effect ofphysiological pH (pH 6.2 to 8) on the initial uptake rate of 20 μM¹⁴C-CPT-11 and 2 μM ¹⁴C-SN-38 was studied. The results summarized inFIG. 6, show that the uptake rate of CPT-11 and SN-38 significantlydecreased by around 65% at a pH greater than 6.8. Alteration of theuptake was also observed when the initial uptake rate of CPT-11 andSN-38 was measured in the presence and absence of bicarbonate. Theuptake of CPT-11 and SN-38 was decreased when the HEPES component of theHank's buffer was replaced by sodium bicarbonate and the pH adjusted togreater than 7.

[0076] Using hamster intestinal cells, the results of the present studyshow that the non-ionic, lactone forms of both CPT-11 and SN-38 wereabsorbed mainly through a passive mechanism but at a respective ratewhich was several times greater than their anionic carboxylate forms(Tables 1, 2 and 3; FIGS. 2, 3 and 4). There were significantdifferences in the transport mechanism as well as in the kineticparameters between jejunal and ileal cells (Tables 2 and 3). Althoughnot shown, similar results were also observed when the uptake of CPT-11and SN-38 was performed using both cecal and colonic cells (26).

[0077] Isolated hamster intestinal cells are not the best model toestimate the cytotoxic effect of SN-38 due to their limited viability toaround 2 hours (Gore et al., 1993). Therefore, HT29 cells were also usedto study the comparative effects of physiological pH on both the initialuptake rate of 2 μM [¹⁴C]SN-38 and the cytotoxicity of 0.4 μM SN-38. Theinitial rate of uptake of SN-38 was lower in HT-29 cells than inisolated hamster intestinal cells.(FIGS. 3 and 4). However, as observedin isolated hamster intestinal cells, the uptake rate of SN-38 in HT29cells was significantly greater at pH 6.2 and 6.8, than at pH 7.4 and8.0 (Kruskal-Wallis test:P=0.008, Dunn's method:p<0.05) (FIG. 7). Thecytotoxicity of SN-38 for HT29 cells was significantly higher at pH 6.2and 6.8 than at pH 7.4 and 8.0 (Kruskal-Wallis test:P=0.007; Dunn'smethod:p<0.05). FIG. 5 shows the relationship between the initial rateof uptake of [¹⁴C]SN-38 and the cytotoxicity of SN-38, indicating that,with decreasing pH, a higher uptake rate correlated with a morecytotoxic effect.

[0078] The results clearly showed that CPT-11 and SN-38 carboxylate weretaken up by the intestinal cells through an active mechanism (Tables 2and 3; FIG. 3). Recently, it has been proposed that cMOAT mostlyexpressed in hepatic canalicular membranes transports several types oforganic anions into the bile as a primary active transport system (24,27-29). Furthermore, the hepatic cMOAT has been reported to beresponsible for the biliary excretion of the anions, SN-38 carboxylate,SN-38-Glu lactone and carboxylate (12, 17). The anion CPT-11 carboxylatewas reported to be only partially eliminated through cMOAT (12, 17). Theinventor's work shows that, in contrast to that of CPT-11 carboxylate,the initial uptake rate of SN-38 carboxylate was significantly inhibitedby DNP-SG and SN-38-Glu (Table 3). These results are in accordance withthose of Cho, et al (12, 17) using hepatic canalicular membranevesicles. Therefore, this work underlines the involvement of a cMOAT orcMOAT-like transporter in jejunal and ileal cellular uptake of SN-38.

[0079] The inventors also reports that CPT-11 lactone and carboxylate,as well as SN-38 lactone can form micelles in the presence of highconcentrations of TCA (FIG. 5). The percentage of the monomerconcentration ranged from 38 to 47%. These concentrations differed fromthose of long-chain fatty acids (i.e. 2.3% for oleic acid) and fromcholesterol (3%). Furthermore, micelle formation inhibited CPT-11uptake, differing from the positive role bile acid micelle formationplays in the intestinal uptake of long-chain fatty acid and cholesterol.These results support data showing that micelle formation inhibited theuptake of short-chain fatty acids, such as palmitic acid.

[0080] As described in FIG. 1, the conversion from CPT-11 and SN-38lactone to carboxylate is pH-driven (11, 12). It has previously beenreported that at pH 7.4, 13% of SN-38 and CPT-11, respectively, were intheir lactone form (30). The present study showed that the initialuptake rate of CPT-11 and Sn-38 was several times greater at acidic pH(pH 6.2 and 6.8) than that at neutral or alkaline pH (pH 7.4 and 8)(FIG. 6). Considering the fact that 1) at acidic pH, the non-ioniclactone form of CPT-11 and SN-38 are transported passively, 2) atneutral/basic pH, the anionic carboxylate form of CPT-11 and SN-38 aremostly absorbed actively, and 3) the uptake rates of both CPT-11 andSn-38 lactone are several times greater than that of their carboxylateform, the mechanism of uptake of CPT-11and SN-38 by intestinal cellsclosely resembles that of short-chain fatty acids. This hypothesis willbe supported by the fact that, as with short-chain fatty acids, micelleformation reduced the uptake of CPT-11 and SN-38 and that the uptake ofCPT-11 and SN-38 is not limited to the small intestine but also takesplace in the cecum and colon.

[0081] Therefore, as for short-chain fatty acids, alkalinization of bileand luminal content reduce the intestinal uptake of CPT-11 and SN-38.The biliary content of CPT-11 and its metabolites was determined for twomen who were treated by cisplatin and received CPT-11 intravenously (9).The major component of the bile was CPT-11 (75.6-91.9%) while SN-38 andSN-38-Glu were minor components, 0.9-3.3% and 7.3-18.9%, respectively.Furthermore, the pH of human bile has been reported to range from 6.5 to8.0 (31). It is therefore, considered that not only the carboxylate butalso lactone the form of CPT-11 plays an important role inpharmacokinetics due to the greater absorption of CPT-11 lactone byintestinal epithelial cells, resulting in an increased level of CPT-11in the enterohepatic circulation.

[0082] SN-38 is active mainly as the lactone form, while SN-38carboxylate exhibits only minor topoisomerase I-inhibitory activity(32). Using rat whole body autoradiography, 24 h after IV injection of¹⁴C-SN-38, the radioactivity was found exclusively in thegastrointestinal tract (33). SN-38 exhibits strong cytotoxicity,SN38-Glu is a deactivated glucuronidated form of SN-38, and CPT-11 ismuch less cytotoxic compared to SN-38 (Kawato et al., 1991).Accumulation of SN-38 in the intestine was shown in rats (Atsumi et al.,1995), and was thought to be responsible for the diarrhea attributed toCPT-11 administration in nude mice (Araki et al., 1993). Disruption ofthe intestinal epithelium in the cecum was observed in mice and ratswith diarrhea after CPT-11 administration (Takatsuna et al., 1996; Ikunoet al., 1995; Araki et al., 1993). The diarrhea induced by CPT-11administration in human was reported to be secretory diarrhea (Bleibergand Cvitkovic, 1996). However, as reported in the animal models, weobserved lethal small-intestinal injury associated to CPT-11-inducedside effects in patients (Kobayashi et al., 1998b).

[0083] Furthermore, accumulation of SN-38, the radioactivity was foundexclusively in the gastrointestinal tract (33). Accumulation of SN-38 inthe intestine was shown to be responsible for the diarrhea attributed toCPT-11 in nude mice (34). Disruption of the intestinal epithelium in thececum was thought to be responsible for CPT-11-induced diarrhea in rat(35). Finally, from clinical estimations in Europe, the diarrhea inducedby CPT-1 was reported to be secretory diarrhea (36), while in our studyapplicants experienced severe incidence of small-intestinal injury (37).

[0084] Autopsy revealed the presence of pseudomembranes jejunoileitis,of which the appearance under light microscopy was characterized by thedisruption of the intestinal epithelium, suggesting that damage diarrheacould occur in severe cases. A mechanism for CPT-11-induced diarrhea isbelieved to include the reabsorption of mainly lactone SN-38 and CPT-11,by the intestinal epithelium, resulting in a high exposure of theintestinal epithelium to these metabolites which causes structural andfunctional injuries to the intestinal tract.

[0085] As suggested in the present study, alkalization of bile and/orintestinal luminal content reduces the uptake of and the exposure of theintestinal epithelium to CPT-11 and SN-38 lactone. The absorption ofshort-chain fatty acids in the intestine has been studied for the pastdecade, and there have been reports of conflicting results. It isbelieved that decreasing pH induces an increased uptake of short-chainfatty acids, as reported in FIG. 6. Thus, a prevention treatment ofcamptothecin and CPT-11-induced diarrhea focuses on two objectives: 1)alkalinization of the intestinal lumen, and 2) clearance of CPT-11 andSN-38 from the body (i.e. stool control). A combination of sodiumbicarbonate, magnesium oxide and water at pH greater than 7 isadministered orally to patients prior and/or simultaneously withstandard IV administration of CPT-11. The incidence of diarrhea isdecreased.

[0086] The relationship between the cellular uptake of SN-38 and itsassociated cytotoxicity was also estimated in the present study. It wasfound that the cellular uptake and cytotoxicity of SN-38 in HT29 cellswas pH-dependent, and that the cytotoxicity correlated well with theinitial uptake rate (FIG. 8). As previously described, it is consideredthat at acidic pH, the predominant form of SN-38 is lactone. This wouldlead to both a greater cellular uptake and intracelluar concentration ofSN-38 lactone. Since SN-38 is active mainly as the lactone form, whileSN-38 carboxylate exhibits only minor topoisomerase I-inhibitoryactivity (Kawato et al., 1991), this should be associated to anincreased cell death. Therefore, one possible mechanism forCPT-11-induced diarrhea might include the reabsorption of SN-38 lactoneby the intestinal epithelium, resulting in structural and functionalinjuries to the intestinal tract.

[0087] In summary, the present study is the first to estimate the uptakeof CPT-11 and SN-38 by intestinal epithelial cells. CPT-11 and SN-38lactone are both passively transported, while both CPT-11 and SN-38carboxylate are actively absorbed. The uptake rate of CPT-11 and SN-38lactone is several times greater than that of the respective carboxylateform. Furthermore, the higher uptake rate of SN-38 is associated with anincreased cytotoxic effect in HT29 cells. These findings suggest thatthe converstion to carboxylate would reduce the cellular uptake of bothCPT-11 and SN-38. Consequently, these findings provide support foralkalization of the intestinal lumen as a possible mechanism to reducereabsorption of CPT-11 and SN-38 in clinical practice. It is possiblethat limited intestinal reabsorption in turn modulates thebioavailability of this drug circulating enterohepatically, and reducesthe toxic side effects of SN-38 on intestinal epithelium.

[0088] The results directly impact clinical practice, and administrationof camptothecin compounds which are cleared through the liver, such asirinotecan hydrochloride and its derivatives. The inventors provide fororal alkalinization with the administration of camptothecin compoundswhich are cleared through the liver, including CPT-11.

[0089] In conclusion, the inventors describe the uptake of camptothecincompounds such as CPT-11 and SN-38 by intestinal epithelium. CPT-11 andSN-38 lactone are both passively transported by intestinal cells. BothCPT-11 and SN-38 carboxylate are actively absorbed, although throughdifferent transport mechanisms. The formation of micelles with TCAreduced the uptake of both CPT-11 and SN-38. The uptake rate of CPT-11and SN-38 lactone is several times greater than that of the carboxylateform while the uptake rate decreased in the presence of bicarbonate andunder condition of increased pH. These findings for CPT-11 and SN-38 canbe useful in clinical practice.

[0090] Table I: Initial rates of uptake of CPT-11 and SN-38 byintestinal cells. jejunum ileum CPT-11

SN-38

[0091] The initial rates of uptake of [¹⁴C]CPT-11 (20 μM) and [¹⁴C]SN-38(2 μM), lactone and carboxylate, respectively, were compared. Theresults are expressed as p mol10⁶ cells⁻¹min⁻¹ and are the mean ±SE of10 experiments. Mann Whitney test was used for statistical analyses.

[0092] Table II: Kinetic Parameters of CPT-11 and SN-38 Uptake byIntestinal Cells TABLE II Kinetic parameters of CPT-11 and SN-38 uptakeby intestinal cells jejunum ileum Km Vmax Kd Km Vmax Kd CPT-11 LactoneND ND 0.95 ND ND 1.06 (0.15) (0.28) Carboxylate 51.3 146.9 <0.05 50.5157.3 <0.05 (16.3) (41.3) (<0.02) (13.0) (38.0) (<0.02) SN-38 Lactone*ND ND 2.38 ND ND 1.87 (0.26) (0.10) Carboxylate* ND ND 0.44 ND ND 0.42(0.17) (0.01)

[0093] (*): Because of limited solubility, only concentrations of SN-38up to 2 μM were investigated.

[0094] (⁺): Because SN-38 carboxylate is judged to be activelytransported from the estimation of its uptake in the presence ofdinitrophenol (Table 3), these values are not considered to bephysiologically relevant.

[0095] NOTE: The data were fitted by least-square nonlinear regressionanalysis using the equation V=(V_(max)S)/(K_(m)+S)+K_(d)S. V_(max) (pmol10⁶ cells⁻¹min⁻¹) is the maximum rate of uptake, K_(m) (μM) is theapparent Michaelis constant, K_(d) (p mole10⁶ cells⁻¹min⁻¹μM⁻¹) isthe rate of diffusion and S (μM) is the concentration of either CPT-11or SN-38. Values are mean ±SE. The major component of the uptake ofCPT-11 lactone, SN-38 lactone and SN-38 carboxylate, respectively, wasnon-saturable and therefore, the K_(m) and V_(max) values were notdetermined (ND).

[0096] Table III: Effect of Dinitrophenol, SN38-Glu and DNP-SG onInitial Uptake Rate of CPT-11 and SN-38 TABLE III Effect ofdinitrophenol, SN38-Glu and DNP-SG on initial uptake rate of CPT-11 andSN-38 CPT-11 SN-38 CPT-11 SN-38 carboxylate carboxylate lactone lactonejejunum ileum jejunum ileum jejunum ileum jejunum ileum Dinitrophenol (1mM) Mean 22.6 29.2 25.5 30.8 94.1 105.5 96.1 134.9 (SE) (13.5) (9.2)(12.4) (13.1) (18.4) (15.6) (14.7) (19.0) P value¹(n = 5) 0.016 0.0080.008 0.016 NS NS NS NS SN38-Glu (200 μM) Mean 108.9 93.9 40.1* 28.9*88.9 NE 54.3 NE (SE) (22.1) (14.3) (11.1) (11.2) (15.3) (20.6) DNP-SG(200 μM) Mean 103.2 105.8 32.0* 28.5* 105.4 NE 78.8 NE (SE) (17.0)(36.3) (9.9) (11.9) (12.7) (24.4) p value² (n = 5) NS NS 0.007 0.020 NSNS # expressed as percentage (%) of control. Differences betweendinitrophenol and its control were evaluated by ¹Mann-Whitney test.Differences among SN-38Glu, DNP-SG and their control were evaluated by²Kruskal-Wallis test, and the significant difference from respectivecontrol was analyzed according to Dunn's method (*p < 0.05).

[0097] Irinotecan Hydrochloride Formulations

[0098] In a preferred embodiment sodium bicarbonate, magnesium oxide andwater are administered at more than a pH of about 7, preferably pH of 8to 10 and most preferably a pH of 8 to 9, provided to patients treatedwith camptothecin compounds such as CPT-11 and its derivatives.

[0099] Further, the CPT-11 compounds of the present invention are usefulin pharmaceutical compositions for systemic administration to humans andanimals in unit dosage forms, such as tablets, capsules, pills, powders,granules, suppositories, sterile parenteral solutions or suspensions,sterile non-parenteral solutions or suspensions oral solutions orsuspensions, oil in water or water in oil emulsions and the like,containing suitable quantities of an active ingredient. For oraladministration either solid or fluid unit dosage forms can be preparedwith the compounds. The compounds are useful in pharmaceuticalcompositions (wt %) of the active ingredient with a carrier or vehiclein the composition in about 1 to 20% and preferably about 5 to 15%.

[0100] Either fluid or solid unit dosage forms can be readily preparedfor oral administration. For example, the CPT-11 can be mixed withconventional ingredients such as dicalciumphosphate, magnesium aluminumsilicate, magnesium stearate, calcium sulfate, starch, talc, lactose,acacia, methyl cellulose and functionally similar materials aspharmaceutical excipients or carriers. A sustained release formulationmay optionally be used. Capsules may be formulated by mixing thecompound with a pharmaceutical diluent which is inert and inserting thismixture into a hard gelatin capsule having the appropriate size. If softcapsules are desired a slurry of the compound with an acceptablevegetable, light petroleum, or other inert oil can be encapsulated bymachine into a gelatin capsule.

[0101] Suspensions, syrups and elixirs may be used for oraladministration of fluid unit dosage forms. A fluid preparation includingoil may be used for oil soluble forms. A vegetable oil such as corn oil,peanut oil or safflower oil, for example, together with flavoringagents, sweeteners and any preservatives produces an acceptable fluidpreparation. A surfactant may be added to water to form a syrup forfluid unit dosages. Hydro-alcoholic pharmaceutical preparations may beused having an acceptable sweetener such as sugar, saccharine or abiological sweetener and a flavoring agent in the form of an elixir.

[0102] Pharmaceutical compositions for parenteral and suppositoryadministration can also be obtained using techniques standard in theart.

[0103] Suitable pharmaceutical carriers include-sterile water; saline,dextrose; dextrose in water or saline; condensation products of castoroil and ethylene oxide combining about 30 to about 35 moles of ethyleneoxide per mole of castor oil; liquid acid; lower alkanols; oils such ascorn oil; peanut oil, sesame oil and the like, with emulsifiers such asmono- or di-glyceride of a fatty acid, or a phosphatide, e.g., lecithin,and the like; glycols; polyalkylene glycols; aqueous media in thepresence of a suspending agent, for example, sodiumcarboxymethylcellulose; sodium alginate; poly(vinylpyrolidone); and thelike, alone, or with suitable dispensing agents such as lecithin;polyoxyethylene stearate; and the like. The carrier may also containadjuvants such as preserving stabilizing, wetting, emulsifying agentsand the like together with the penetration enhancer of this invention.

[0104] The effective dosage for mammals may vary due to such factors asage, weight activity level or condition of the subject being treated.Typically, an effective dosage of a compound according to the presentinvention is about 10 mg/m² to 700 mg/m² when administered by eitheroral or rectal dose from 1 to 3 times daily. CPT-11 may preferably beadministered once a week for a 1 to 5 week period. Administration timesand dosages of CPT-11 for the treatment of cancers and tumors are known.

[0105] Administration of Irinotecan Hydrochloride

[0106] The mean terminal elimination half-life of irinotecanhydrochloride (Pharmacia-Upjohn) is about 6 hours.

[0107] Camptothecin compounds may also be administered alone or incombination with combination chemotherapy regimens including leucovorin,cisplatin, 5-FU, oxiplatin as well as other known chemotherapeutics. Inan alternative embodiment camptothecin compounds such as irinotecanhydrochloride may also be administered with loperamide. camptothecincompounds such as irinotecan hydrochloride may also be administered withloperamide.

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[0171] The purpose of the above description and examples is toillustrate some embodiments of the present invention without implyingany limitation. It will be apparent to those of skill in the art thatvarious modifications and variations may be made to the composition andmethod of the present invention without departing from the spirit orscope of the invention. All patents and publications cited herein areincorporated by reference in their entireties.

1. A method of inhibiting a diarrhea side effect of camptothecincompounds comprising administering said camptothecin compounds at analkaline pH.
 2. The method of claim 1, wherein the camptothecincompounds are selected from the group consisting of irinotecanhydrochloride (CPT-11), SN-38-Glu, SN-38 and its derivatives.
 3. Themethod of claim 1, wherein the intestinal lumen and the bile ismaintained at an alkaline pH.
 4. The method of claim 1, wherein theirinotecan hydrochloride is administered intravenously, orally orintramuscularly.
 5. The method of claim 1, wherein reabsorption of saidcamptothecin compounds by the intestines is inhibited.
 6. A method oftreating cancer comprising administering irinotecan hydrochloride andits derivatives while maintaining the intestinal lumen at an alkalinepH.
 7. The method of claim 6, wherein the cancer is selected from thegroup consisting of breast cancer, ovarian cancer, colon cancer,malignant melanoma, small cell lung cancer, thyroid cancers, lymphomasand leukemias.
 8. The method of claim 6, wherein the alkaline pH is a pHfrom 7 to
 10. 9. The method of claim 6, wherein the alkaline pH is a pHfrom
 7. 10. The method of claim 6, wherein the alkaline pH is a pH from8.
 10. The method of claim 5, wherein the alkaline pH is a pH from 9.11. The method of claim 5, wherein the alkaline pH is a pH from
 10. 12.A method of treating AIDS comprising administering irinotecanhydrochloride or its derivatives while maintaining the intestinal lumenat an alkaline pH.
 13. A method of treating cancer comprisingadministering a compound selected from the group consisting of 7-hydroxy-methyl camptothecin, irinotecan hydrochloride,aminocamptothecin, DX-8951F, SN-38, HAR4, HAR5, HAR6, HAR7, HAR8 andtopotecan, while maintaining the intestinal lumen at an alkaline pH. 14.A kit comprising a pharmaceutical composition including irinotecanhydrochloride (CPT-11) in combination with a suitable amount ofbicarbonate to maintain the intestinal lumen at an alkaline pH.
 15. Thecomposition of claim 14, wherein the bicarbonate is selected from thegroup consisting of sodium bicarbonate, magnesium bicarbonate, potassiumbicarbonate and mixtures thereof.
 16. A method of administering acamptothecin compound comprising prior to or simultaneouslyadministering said camptothecin compound, orally administering acomposition comprising borbic acid.
 17. A method of administering acamptothecin compound comprising prior to or simultaneouslyadministering said camptothecin compound, orally administering acomposition comprising urso-deoxycholic acid.